The present invention is generally related to polynucleotide molecules and to their use for production of desired products after introduction thereof into human or animal cells.
More specifically, the present invention is directed to alphavirus expression vectors comprising at least part of an alphavirus genome and heterologous RNA inserted downstream of an alphavirus base sequence having translation enhancing activity. Such vectors can be used to achieve enhanced levels of expression of DNA or cDNA coding for a desired product and being complementary to said heterologous RNA after introduction of said vector in eukaryotic cells in cell culture or in a living body.
The modern techniques in molecular biology have opened up many completely unforeseen possibilities for the pharmaceutical and biotechnological industry. For instance, it is today possible to clone any gene (once identified) as a DNA (or cDNA) molecule and express it in prokaryotic and/or eukaryotic cells. This has facilitated the production of the corresponding proteins in vitro for therapeutic use and/or prophylactic use, e.g. as a vaccine. Lately the possibilities of gene expression technology have been extended also to in vivo use in whole animals and even in human beings. Illustrative of this recent development are human gene therapy (1) and genetic immunization (2). In order to express a foreign gene in cells in vivo or in vitro, the gene segment, or the corresponding cDNA, comprising the coding sequences, is usually inserted into a so called expression vector. This vector provides for all elements that are necessary for the transcription and translation of the gene, or the cDNA thereof, within the cell. Today, there exist very efficient expression vectors for bacterial and yeast cells. However, this is not the case for animal, such as mammalian, cells. This creates a large problem when a protein with mammalian specific-modifications has to be produced and isolated for a therapeutic purpose or for prevention of disease or in cases where expression in whole organisms, e.g. the living body of an animal or human, is required. Therefore, in very general terms there is a great demand for new expression vectors for use in animal, e.g. mammalian, cells, which vectors have
(i) increased protein expression efficiency,
(ii) broadened host cell specificity, and
(iii) increased safety.
Recently, a completely new type of DNA expression vectors have been developed for use in animal, e.g. mammalian, cells. These vectors are based on the alphavirus genome.
Alphavirus is a genus belonging to the family Togaviridae having single stranded RNA genomes of positive polarity enclosed in a nucleocapsid surrounded by an envelope containing viral spike proteins.
The Alphavirus genus comprises among others the Sindbis virus, the Semliki Forest virus (SFV), the Ross River virus and Venezuelar, Western and Eastern equine encephalitis viruses, which are all closely related. In particular, the Sindbis and the Semliki Forest viruses have been widely studied and the life cycle, mode of replication, etc, of these viruses are well known and thus, need not to be specifically discussed herein.
Alphaviruses replicate very efficiently in animal cells which makes them valuable as vectors for production of protein and nucleic acids in such cells.
Expression systems based on the Sindbis virus are disclosed in U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879. The Sindbis virus vectors of U.S. Pat. No. 5,091,309 comprise RNA derived from Sindbis defective interfering (DI) RNA having heterologous RNA inserted therein. In U.S. Pat. No. 5,217,879 self-replicating and self-packaging recombinant Sindbis virus RNA molecules are disclosed comprising a heterologous coding sequence and at least one Sindbis virus junction region able to direct Sindbis virus subgenomic messenger RNA synthesis in a host cell. RNA transcripts are synthesized in vitro by transcription of Sindbis virus cDNA which has been inserted in a plasmid under control of a promoter, such as SP6.
Xiong At al., Science, Vol 243, 1989, 1188-1191(3) also disclose a gene expression system based on Sindbis virus. This system is said to be efficient in a broad range of animal cells. Expression of the bacterial CAT (chloramphenicol acetyltransferase) gene in insect, avian and mammalian cells inclusive of human cells is disclosed therein.
In Bio/Technology, Volume 9, pages 1356-1361, 1991(4), Liljestrxc3x6m and Garoff disclose animal cell expression vectors based on the SFV replicon. When foreign DNA coding sequences are inserted into these vectors, high amounts of foreign protein are obtained.
According to WO 92/10578, an RNA molecule is provided, which is derived from an alphavirus RNA genome and is capable of efficient infection of animal cells, which RNA molecule comprises the complete alphavirus genome regions, which are essential for replication of the said alphavirus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof. According to WO 92/10578 such RNA molecules can be transferred into animal cells by any means of transfection or by packaging of said RNA molecules into infectious alphavirus particles for later infection of animal cells. In both cases the transfected or infected RNA molecule will be able to replicate within the target animal cell and to express the exogenous RNA sequences inserted into said RNA molecule. Such molecules and strategies for their expression within the cell can be used as vaccines or strategies to vaccinate in order to prevent or treat infection or cancer. In this reference, SFV has been used to illustrate alphaviruses.
The above mentioned expression vectors based on the alphavirus genome have been shown to promote a higher protein expression efficiency than earlier mammalian protein expression systems. They have also been shown to work in almost all higher eukaryotic cell types. Furthermore, they have been complemented with highly stringent safety features to prevent spreading of the virus(5). Important proteins, to be used for prevention of disease, like the HIV spike protein, have been produced with this system and such proteins have been shown to have a more native-like structure than when produced in other systems(6). Alphavirus vectors have also been used successfully for genetic immunization(7).
The present invention is directed to a significant and unforeseen improvement of the alphavirus expression vectors.
More specifically, in accordance with the present invention it has been found that there are nucleotide base sequences within the alphavirus genome, which sequences have translation enhancing activity. In comparison to the expression levels of prior known alphavirus vectors, the level of expression of a desired substance which can be obtained according to the present invention is increased about 10-fold.
Thus, the present invention is generally directed to expression of heterologous DNA in eukaryotic cells, alphavirus vectors comprising at least part of an alphavirus genome being used to achieve expression, said vectors further comprising RNA complementary to the heterologous DNA inserted essentially immediately downstream of a nucleotide base sequence having translation enhancing activity.
According to the present invention, such base sequences having translation enhancing activity (also designated translational enhancers) comprise a 5xe2x80x2 portion of an alphavirus capsid gene or the complete capsid gene. Suitably, this translational enhancer is endogenous to the alphavirus vector. However, exogenous translational enhancers of another alphavirus species origin might be used, at least occasionally.
Thus, the present invention is also related to a self-replicative and transcription competent recombinant alphavirus RNA molecule comprising at least part of an alphavirus RNA genome and heterologous RNA encoding a substance having biological activity located downstream of an alphavirus base sequence having translation enhancing activity, said translation enhancing base sequence being comprised of a complete alphavirus capsid gene or a 5xe2x80x2 portion of said gene.
A suitable embodiment of the present invention is concerned with expression vectors based on the Semliki Forest virus (SFV) genome. The complete SFV capsid gene encodes 267 aminoacid residues, and, thus, comprises 801 bases.
For SFV, a translation enhancing activity has been found to reside in the first 102 bases of this capsid gene, said activity giving rise to a protein production at a level of about 85% of the wild-type capsid protein. This is an about 10-fold increase in comparison to the prior known SFV vectors.
A sequence of said gene comprising the first 81 bases also gives rise to an increased level of expression, although to a less extent.
Thus, the present SFV translational enhancer comprises at least the first 81 bases, and preferably at least the first 102 bases of the capsid gene and at most 801 bases, i.e. a base sequence corresponding to the complete capsid gene.
The sequence of the said 5xe2x80x2 portion of the SFV capsid gene comprised of the first 102 bases, reading from 5xe2x80x2 to 3xe2x80x2 end, is ATGAATTACA TCCCTACGCA AACGTTTTAC GGCCGCCGGT GGCGCCCGCG CCCGGCGGCC CGTCCTTGGC CGTTGCAGGC CACTCCGGTG GCTCCCGTCG TC (SEQ ID NO:1).
Modifications of this base sequence (e.g. base deletions, substitutions, and/or additions) having an essentially conserved enhancing activity are also encompassed by the present invention.
In the following illustrative Examples the occurrence of a translational enhancer has been demonstrated for SFV. In view of the homology between various alphavirus species, it can be expected that a similar mechanism of translation enhancement exists in all alphaviruses. However, as the sequence between different alphaviruses vary considerably in the 5xe2x80x2 end of the C gene region, it is most likely that some similarities in the features of the secondary and the tertiary structure of the RNA molecule are responsible for the translation enhancing effect. This means that the exact sequences and probably also the length of the translation enhancing regions will vary between different alphaviruses.
The present invention is also related to a DNA molecule comprising DNA sequences complementary to the present recombinant alphavirus RNA molecule, which DNA molecule also may comprise a DNA sequence encoding a promoter, such as SP6, for transcribing the recombinant RNA molecule in cells and additional DNA sequences encoding traits required for plasmid growth in E. coli. 
The present alphavirus vectors having enhanced translation capacity can be used for the same purposes as the previously known alphavirus vectors. Thus, they are likely to be very useful for production of substances having biological activities, such as proteins or polypeptides, in eukaryotic cells, especially mammalian ones, which substances can be used for biotechnical or medical purposes. For instance, the heterologous RNA may suitably encode a protein, polypeptide or peptide having therapeutic activity or prophylactic, such as immunogenic or antigenic, activity. Said eukaryotic cells, wherein expression is achieved, can be available as cell cultures or constitute a part of a living organism, such as an animal or human being. The present invention is also concerned with cells transformed with the present recombinant RNA or DNA molecule and cell lines stably transformed with said RNA or DNA molecule.
As stated above, the alphavirus expression vectors are based on the alphavirus genome. This consists of a single-stranded RNA molecule of positive polarity. In the infected cell the 5xe2x80x2 ⅔ of this RNA serves as a mRNA for the viral nonstructural polyprotein, which is co- and posttranslationally cleaved into four mature proteins(8, 9). These proteins form the viral polymerase complex which replicates the genomic RNA via an intermediate of negative polarity(10). This intermediate also functions as a template for the synthesis of a subgenomic RNA molecule, which is colinear with the 3xe2x80x2 ⅓ of the viral genome. This subgenomic transcript (also called 26S mRNA) is translated into the viral structural poly-protein. Processing of this polyprotein is initiated by the amino terminal capsid (C) protein, which autocatalytically cleaves itself from the nascent polyprotein chain(11, 12, 13, 14). The remaining part of the structural polyprotein is co-translationally. inserted into the membrane of the endoplasmic reticulum and the spike proteins p62 and E1 are released by signal peptidase-mediated cleavage events(15, 16, 17).
The alphavirus expression vectors are suitably con-structed from a modified cDNA copy of the viral genome, from which at least part of the structural polyprotein-encoding region has been deleted and replaced with a cloning site(3, 4). Heterologous cDNA can be inserted into this site and the corresponding recombinant alphavirus genome can be produced by in vitro transcription. When transfected into host cells the recombinant genome is replicated in a wild-type manner since it contains both the non-structural coding region of the alphavirus and the 5xe2x80x2 and 3xe2x80x2 replication signals. However, instead of the virus structural proteins the subgenomic RNA now directs the synthesis of the heterologous protein. The Semliki Forest virus (SFV) expression system(4) has also been supplemented with an in vivo packaging system whereby recombinant genomes can be packaged into infectious SFV-like particles following co-transfection with a packaging-deficient helper genome, which provides the viral structural proteins(4, 5). These recombinant particles can be used to infect cells either in vivo or in vitro. The host cell range of the recombinant particles is determined by the alphavirus spike and is therefore very broad. The infected cells will produce recombinant genomes and also high amounts of the heterologous proteins. However, as no virus structural proteins are encoded by the recombinant genomes, no new virus particles will be made, and hence there is no spreading of virus.
Accordingly, one embodiment of the present invention is directed to a method to produce a recombinant alphavirus comprising a recombinant alphavirus RNA genome surrounded by a wild-type alphavirus coat, the said RNA genome comprising the present recombinant RNA molecule, by co-transformation, such cotransfection, of cells with the said recombinant RNA genome and a helper RNA containing expression capacity of alphavirus structural proteins and comprising coding sequences for the alphavirus structural proteins, cis acting replication signals but no encapsidation signals, incubation of the cells and collection of medium containing infectious recombinant alphavirus particles.
The present invention is also related to these co-transformed cells producing the infectious particles and to the infectious particles per se.
As indicated above, a broad range of host cells of animal (including human) origin can be used according to the present invention. Such host cells can be selected from avian, mammalian, amphibian, insect and fish cells. Illustrative of mammalian cells are human, monkey, hamster, mouse and porcine cells. Suitable avian cells are chicken cells.
Furthermore, the present invention can be used both in vitro and in vivo. As per definition, in vitro means a process performed outside a living organism as opposed to in vivo which means that a process is performed inside a living organism.
In accordance with the present invention xe2x80x9ctransformationxe2x80x9d is intended to mean introduction in general of exogenous polynucleotides sequences into the interior of a cell, eukaryotic or prokaryotic, and the exogenous polynucleotide sequence may remain in cell cytoplasm, in nucleus as extrachromosomal (episomal) or may be stably integrated into the cell genome. The mode of transformation is not crucial, but any means, known at present or that may be developed in the future, can be used according to the invention.
A further embodiment of the invention is related to a method for expressing a heterologous RNA sequence encoding a biologically active substance comprising infection of cells in cell culture or in an animal or human individual with the present infectious recombinant alphavirus particles.
Suitably, such expression, when achieved in an animal, will not give rise to any effect beneficial to said animal, but the expression product produced in said animal can be recovered from said animal, e.g. in a body fluid, such as blood, milk or ascites.
The present invention is also related to a pharmaceutical preparations comprising the present recombinant RNA in a physiologically administrable form. Such preparations may comprise the present co-transformed cells producing infectious recombinant alphavirus particles or such infectious particles per se.
In the following experimental part, the Semliki Forest virus (SFV) has been used to illustrate the present invention. The earlier described SFV expression system provides three different plasmid vectors (pSFV1-3) with a cloning site for heterologous DNA in, or immediately after, the 5xe2x80x2 untranslated leader sequence of the subgenomic 26S RNA(4). In Example 2 the expression levels of several different heterologous proteins are compared when using the earlier SFV vectors and when using a new SFV vector in which the complete 26S RNA leader plus the complete C protein gene precedes the heterologous protein gene (=the SFVC-vector). It is evident that heterologous products are produced from SFVC-vectors at a substantially higher level than from the earlier vectors and that all C-fusion proteins have been cleaved into separate authentic products via the autoprotease activity of the C protein (FIG. 2). Quantitation shows that there is about a ten-fold difference in expression level.
In example 3 we have used E. coli LacZ (xcex2-galac-tosidase) gene fusions in order to study where in the C gene the enhancing effect resides. Specifically, various portions of the 5xe2x80x2 part of the C gene have been fused to the second codon of the LacZ gene. Expression of these recombinant constructions show that the enhancing effect is located within the 102 first bases of the C gene.
In example 4 we have analyzed whether the effect is expressed on the level of RNA replication and transcription. The production of genomic RNA and subgenomic RNA was compared in high and low producer variants of the LacZ recombinants described in example 3. No differences in RNA levels were found and hence the enhancing effect of the C gene segment must reside on the translational level. This segment most likely contains a specific RNA structure that enhances translational initiation.